Reversible nanoparticle aggregates and methods for detecting proteases

Abstract

In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for detecting proteases including detecting proteases in a biological sample. In alternative embodiments, products of manufacture, formulations, mixtures or kits are used for stabilizing (or substantially stabilizing) nanoparticles in a reversible aggregate, and, detecting the presence of a protease in a fluid, wherein optionally the products formulations or mixtures can aggregate or assemble into nanoparticles such that the nanoparticles undergo plasmonic coupling, and the plasmonic coupling is reversible (or substantially reversible) leading to monodisperse nanoparticles when a chemical cue or signal is added.

1 : A product of manufacture, formulation, mixture or kit for: stabilizing or substantially stabilizing nanoparticles in a reversible aggregate, and, detecting the presence of a protease in a fluid,
 the products of manufacture, formulations, mixtures or kits comprising:   (a) a compound X capable of making or forming into reversibly aggregated (or substantially reversibly aggregated) nanoparticles (NPs),   wherein the compound X comprises:   a nanoparticle (NP) aggregated with or stabilized with (Arginine) x  (or Arg x  or R x ), or an Arg x  citrate-stabilized nanoparticle, or a plurality of Arg x  citrate-stabilized nanoparticles (NPs), or Arg x -NPs, wherein x is an integer 2, 3, 4, 5 or 6, and optionally the R x  comprises AA, AAA, AAAA (SEQ ID NO:1), AAAAA (SEQ ID NO:2) or AAAAAA (SEQ ID NO:3),   a nanoparticle (NP) aggregated with or stabilized with Lysine x -R x  (or -Lys x -R x , or K x -R x , or an K x -R x  citrate-stabilized nanoparticle, or a plurality of K x -R x  citrate-stabilized nanoparticles (NPs), or K x -R x -NPs, wherein x is an integer 1, 2, 3, 4, 5 or 6,   and compound X aggregates (or is capable of aggregating, or substantially aggregates) when in a liquid solution,   (b) a compound Z conjugated to a poly(ethylene glycol) (Z-PEG x ), or a plurality of poly(ethylene glycol) (Z-PEG x s), wherein X is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13,14 or 15, or X is an integer between about 1 and 40, or between about 2 and 30, or compound Z is conjugated to a peptide,   wherein compound Z conjugated to:   (i) a poly(ethylene glycol) (Z-PEG x ), or a plurality of poly(ethylene glycol) (Z-PEG x s) is capable of dissociating compound X aggregates (for example, Arg-Arg-NPs aggregates) in a liquid solution,   wherein each Z-PEG x  is conjugated to a protease peptide substrate or an amino acid sequence specifically recognized and cleaved by a protease by a compound Y comprising a moiety or a functional group linking the Z-PEG x  to the protease peptide substrate (or Z-PEG x -Y-protease peptide substrate), or   (ii) a peptide.   
     
     
         2 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein the protease is a viral or a mammalian protease. 
     
     
         3 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein the fluid comprises or is derived from: (a) a biological fluid, optionally a biological fluid from an in vivo source, optionally an undiluted and/or untreated biological fluid from an in vivo source, and optionally the biological fluid from an in vivo source comprises blood, plasma, saliva, urine, bile, a lacrimal duct solution or tear, or cerebrospinal fluid (CSF); (b) a cell lysate; or (c) water, distilled water, saline or sea water. 
     
     
         4 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein the citrate-stabilized NPs is about 20 nm in diameter, or is between about 10 and 50 nm in diameter. 
     
     
         5 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein the citrate-stabilized NPs is prepared using a Turkevich method comprising:
 (a) rapidly injecting an aqueous solution of sodium citrate tribasic dihydrate (SCTD) (optionally 150 mg SCTD, 5 mL aqueous solution) into an aqueous solution of HAuCl 4 ·3H 2 O (optionally 45 mg HAuCl 4 ·3H 2 O, 300 mL aqueous solution) under boiling conditions and vigorous stirring, to produce a reaction mixture; and   (b) the reaction mixture is left boiling while stirring for another about 15 min and then cooled down to room temperature (RT) to generate a deep red dispersion, and the deep red dispersion was then purified by applying one round of centrifugation at about 18,000 g for about 30 min to generate a pellet of AuNPs-citrate, and the pink supernatant is discarded, and the resulting pellet of AuNPs-citrate is redispersed in deionized water by sonication and stored at ambient conditions.   
     
     
         6 : A solution or formulation comprising the plurality of Arg-Arg-NPs of  claim 1 (a) and the plurality of Z-PEG x -Y-peptide of  claim 1 (b), and optionally the solution or formulation comprises a saline solution, water, a cell lysate or a biological solution, and optionally the biological solution comprises a biological fluid from an in vivo source comprises a cell lysate solution, blood, plasma, saliva, urine, bile, a lacrimal duct solution or tear, or cerebrospinal fluid (CSF). 
     
     
         7 : A method for detecting a protease in a sample, comprising: mixing the Arg-Arg-NPs of  claim 1 (a) and the Z-PEG x -Y-peptide of  claim 1 (b) in a sample, and presence of a protease capable of specifically recognizing and cleaving the peptide is detected via a proteolytic cleavage that releases Z-PEG x  fragments from a Z-PEG-Y-peptide, thus inducing the dissociation of the AuNPs-citrate assemblies and turning the solution from blue to red. 
     
     
         8 : The method of  claim 7 , wherein the sample comprises a cell lysate or a biological solution. 
     
     
         9 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein the products formulations or mixtures can aggregate or assemble into nanoparticles such that the nanoparticles undergo plasmonic coupling, and the plasmonic coupling is reversible, or substantially reversible, leading to monodisperse nanoparticles when a chemical cue or signal is added. 
     
     
         10 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein the NP comprises a di-arginine (Arg) citrate-stabilized nanoparticle, or a plurality of di-arginine (Arg) citrate-stabilized nanoparticles (NPs), or Arg-Arg-NPs. 
     
     
         11 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein the K x -R x  comprises KR, RK, RRK, KRR, RKR, RRKR (SEQ ID NO:4), RKRR (SEQ ID NO:5), KRRR (SEQ ID NO:6), RRRK (SEQ ID NO:7), KKRR (SEQ ID NO:8), KKKR (SEQ ID NO:9), RKKK (SEQ ID NO:10), KRRK (SEQ ID NO:11) or RKKR (SEQ ID NO:12). 
     
     
         12 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein the biological fluid is derived or isolated from an in vivo source. 
     
     
         13 : The product of manufacture, formulation, mixture or kit of  claim 12 , wherein the biological fluid is an undiluted and/or untreated biological fluid from an in vivo source. 
     
     
         14 : The product of manufacture, formulation, mixture or kit of  claim 12 , wherein the biological fluid from an in vivo source comprises blood, plasma, saliva, urine, bile, a lacrimal duct solution or a tear, or cerebrospinal fluid (CSF); a cell lysate; or water, distilled water, saline or sea water, 
     
     
         15 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein compound X, or the citrate-stabilized compound X, or the citrate-stabilized nanoparticle (NP) comprises or is conjugated to a metal to generate a metal-nanoparticle or metal-compound X. 
     
     
         16 : The product of manufacture, formulation, mixture or kit of  claim 15 , wherein the metal of the metal nanoparticle or metal-compound X comprises silver (Ag) (for example, the nanoparticle comprises Arg-Arg-Ag-NP) or gold (Au) (for example, the nanoparticle comprises Arg-Arg-Au-NP). 
     
     
         17 : The product of manufacture, formulation, mixture or kit of  claim 1 , wherein:
 (a) each peptide comprises two three four, five, six, seven, eight, nine, ten, eleven or twelve or more amino acids, and optionally the peptide comprises EEKKPPC (SEQ ID NO:18),   (b) one way of coupling Z to the nanoparticle comprises use of thiol on a Cys moiety,   (c) the peptide comprises one or more amino acids with a negative charge at the opposite end that is bound to the nanoparticle where one or more of these negative amino acids are acetylated to increase negative charge,   (d) each peptide comprises one or more spacer amino acids including glycine, proline or alanine that increase the distance between the binding amino acid and the charged amino acid,   (e) each peptide comprises EEKKPPC (SEQ ID NO:18), where the cysteine (or C) binds to a gold (Au) surface, E (glutamic acid) has a negative charge and is acetylated, K (lysine) is positively charged, and P (proline) adds steric bulk,   (f) the compound Z comprises:
 a thiol to generate a thiolated (HS) poly(ethylene glycol) (HS-PEG x ), or a plurality of thiolated (HS) poly(ethylene glycol) (HS-PEG x s), 
 an alkyne, to generate a poly(ethylene glycol) (ALK-PEG x ), or a plurality of poly(ethylene glycol) (ALK-PEG x s), 
 a lipoic acid group, to generate a poly(ethylene glycol) (LIP-PEG x ), or a plurality of poly(ethylene glycol) (LIP-PEG x s), 
   (g) the compound Y comprises:
 a carboxyl group (for formation of an amide bond between the PEG x  and peptide), 
 an azido group (for formation of a copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) to chemically join the PEG x  and peptide), 
 an alkyne group (for formation of a copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) to chemically join the PEG x  and peptide), or 
 a maleimide group (to generate a Michael reaction addition thiol group to chemically join the PEG x  and peptide). 
   (h) each polyethylene glycol (PEG) moiety, or x, comprises one, two three four, five, six, seven, eight, nine, ten, eleven or twelve or more PEG repetitions,   (i) each Z-PEG x  is conjugated to a protease peptide substrate (or an amino acid sequence specifically recognized and cleaved by a protease) (Z-PEG x -Y-peptide), and/or   (j) a plurality of compound X make or form into reversibly aggregated nanoparticles (NPs) when in the liquid solution, and optionally can stay in a substantially aggregated state indefinitely before being resuspended back to a state of monodispersity by the addition of compound Z to the liquid solution.   
     
     
         18 : The product of manufacture, formulation, mixture or kit of  claim 2 , wherein the viral protease is a coronavirus protease. 
     
     
         19 : The product of manufacture, formulation, mixture or kit of  claim 18 , wherein the coronavirus protease is a Covid-19 protease or SARS-CoV-2 (Mpro). 
     
     
         20 : The method of  claim 8 , wherein the biological solution comprises a biological fluid from an in vivo source comprises a cell lysate solution, blood, plasma, a lacrimal duct solution or (a tear), saliva, urine, bile or cerebrospinal fluid (CSF); or the sample comprises water or a saline solution.